Many peptides and proteins (collectively referred to herein as "polypeptides") are potentially useful as therapeutic agents but lack an adequate method of administration.
The usefulness of polypeptides as therapeutic agents is limited by the biological barriers that must be traversed before a polypeptide can reach its specific in vivo target. Parenterally administered polypeptides are readily metabolized by plasma proteases. Oral administration, which is perhaps the most attractive route of administration, is even more problematic. In the stomach, orally administered polypeptides risk enzymatic proteolysis and acidic degradation. Survival in the intestine is even more unlikely due to excessive proteolysis. In the lumen, polypeptides are continuously barraged by a variety of enzymes, including gastric and pancreatic enzymes, exo- and endopeptidases, and brush border peptidases. As a result, passage of polypeptides from the lumen into the bloodstream is severely limited.
There is therefore a need in the art for means which enable parenteral and oral administration of therapeutic polypeptides.
2.1 Routes of Administration of Polypeptide Drugs
The problems associated with oral and parenteral administration of polypeptides are well known in the pharmaceutical industry. Various strategies have been used in attempts to improve oral and parenteral delivery of polypeptides.
Penetration enhancers (e.g., salicylates, lipid-bile salt-mixed micelles, glycerides, and acylcarnitines) has been investigated for improving oral administration. However, penetration enhancers frequently cause serious local toxicity problems, such as local irritation and toxicity, partial or complete abrasion of the epithelial layer, as well as tissue inflammation. Furthermore, penetration enhancers are usually co-administered with the polypeptide drug, and leakages from the dosage form are common.
Another common strategy for enhancing oral delivery is co-administration of the polypeptide drug with a protease inhibitor (e.g., aprotinin, soybean trypsin inhibitor, and amastatin). Unfortunately, protease inhibitors also inhibit the desirable effects of proteases. Accordingly, methods and compositions are needed for effectively delivering polypeptide drugs in the absence of protease inhibitors.
Attempts have also been undertaken to modify the physiochemical properties of polypeptide drugs to enhance penetration of such drugs across mucosal membranes. One such approach has been to conjugate polypeptide drugs to lipophilic molecules; however, results have suggested that simply raising lipophilicity is not sufficient to increase paracellular transport.
Other methods for stabilizing polypeptides have been described. Thus, for example, Abuchowski and Davis have disclosed various methods for derivatizating enzymes to provide water-soluble, non-immunogenic, in vivo stabilized products ("Soluble polymers-Enzyme adducts", Enzymes as Drugs, Eds. Holcenberg and Roberts, J. Wiley and Sons, New York, N.Y., (1981)). Abuchowski and Davis disclose various ways of conjugating enzymes with polymeric materials, such as dextrans, polyvinyl pyrrolidones, glycopeptides, polyethylene glycol and polyamino acids. The resulting conjugated polypeptides are reported to retain their biological activities and solubility in water for parenteral applications. Furthermore, U.S. Pat. No. 4,179,337 discloses that polyethylene glycol renders proteins soluble and non-immunogenic. However, these polymeric materials do not contain components which improve intestinal mucosa binding or which facilitate or enhance membrane penetration. Thus, these conjugates are not intended for oral administration.
Meisner et al., U.S. Pat. No. 4,585,754, teaches that proteins may be stabilized by conjugating them with chondroitin sulfates. Products of this combination are usually polyanionic, very hydrophilic, and lack cell penetration capability; they are usually not intended for oral administration.
Mill et al., U. S. Pat. 4,003,792, teaches that certain acidic polysaccharides, such as pectin, algesic acid, hyaluronic acid and carrageenan, can be coupled to proteins to produce both soluble and insoluble products. Such polysaccharides lack the capacity to improve cell penetration characteristics and are not intended for oral administration.
Other researchers have shown that polyethylene glycol linked to a protein improves stability against denaturation and enzymatic digestion. (Boccu et al. Pharmacological Research Communication 14, 11-120 (1982)). However, these polymers do not contain components for enhancing membrane interaction. Thus, the resulting conjugates suffer from the same problems as noted above and are not suitable for oral administration.
Conjugation of polypeptides to low molecular weight compounds (e.g., aminolethicin, fatty acids, vitamin B12, and glycosides) has also been described (R. Igarishi et al., "Proceed. Intern. Symp. Control. Rel. Bioact. Materials, 17, 366, (1990); T. Taniguchi et al. Ibid 19, 104, (1992); G. J. Russel-Jones, Ibid, 19, 102, (1992); M. Baudys et al., Ibid, 19, 210, (1992)). The resulting polymers do not contain components necessary to impart both solubility and membrane affinity necessary for bioavailability following oral administration.
Encapsulation of proteinaceous drugs in an azopolymer film has also been employed as a means for enabling oral administration of polypeptide drugs (M. Saffan et al., in Science, 223, 1081, (1986)). The film is reported to survive digestion in the stomach but is degraded by microflora in the large intestine where the encapsulated protein is released. This approach is also known to lengthen the in vivo duration of action of polypeptide drug. However, the technique utilizes a physical mixture and does not facilitate the absorption of released protein across the membrane.
Similarly, liposomes have been used to stabilize polypeptide drug for oral as well as parenteral administration. A review of the use of liposomes is found in Y. W. Chien, "New Drug Delivery Systems", Marcel Dekker, New York, N.Y., 1992. Liposome-protein complexes are physical mixtures. Results of liposome-based administration are often erratic and unpredictable. Furthermore, use of liposomes can result in undesirable accumulation of the polypeptide drug in certain organs. Other disadvantages of liposome-based formulations include high cost, complex manufacturing processes requiring complex lypophilization cycles, and solvent incompatibilities.
Another approach for facilitating the oral delivery of polypeptide drugs is the use of "proteinoids" (Santiago, N. et al. "Oral Immunization of Rats with Influenza Virus M Protein (M1) Microspheres", Abstract#A 221, Proc. Int. Symp. Control. Rel. Bioac. Mater.,19, 116 (1992)). Protenoids encapsulate the drug of interest in a polymeric sheath composed of highly branched amino acids. As with liposomes, the polypeptide drugs are not chemically bound to the proteinoid sphere; leakage of drug components from the dosage form is possible.
Attempts have been made to use emulsions as matrices for drug delivery of labile drugs (e.g., drugs such as insulin, which are susceptible to enzymatic, chemical, or physical degradation). However, in spite of preliminary reports on the efficacy of emulsion formulations in promoting the intestinal absorption of insulin in rats and rabbits (see Engel, S. et al., "Insulin: intestinal absorbtion as water-in-will-in-water emulsions," Nature, 219:856-857 (1968); Shichiri, Y. et al., "Enteral absorption of water-in-oil-in-water insulin emulsions and rabbits," Diabetologia, 10: 317-321 (1974)), subsequent research was abandoned because of the lability of the insulin and the need for excessive doses to maintain glucose homeostasis (Shichiri, Y. et al., "Increased intestinal absorbtion of insulin: an insulin suppository," J. Pharm. Pharamcol., 30:806-808 (1978); Block, L. et al. "Pharmaceutical Emulsions and Microemulsions," Pharmaceutical Dosage Forms: Disperse Systems, Vol. 2, p. 71 (1996)). Therefore, there remains a needed the art for methods and compositions which enable the use of emulsions and microemulsions for delivering labile drugs, such as insulin.
There is clearly a need in the art for means which (1) enable polypeptide drugs to survive in the gut and penetrate the gut epithelium to enter the bloodstream; (2) enable polypeptide drugs to survive in the bloodstream in an active form, and (3) provide polypeptide drugs having a delayed onset of action, and/or increased duration of action. The present invention provides means for solving each of these three important problems.
2.2 Diabetes and Insulin
Diabetes, a disorder of carbohydrate metabolism, has been known since antiquity. Diabetes results from insufficient production of or reduced sensitivity to insulin. Insulin is synthesized in the beta cells of the islets of Langerhans of the pancreas and is necessary for normal utilization of glucose by most cells in the body. In persons with diabetes, the normal ability to use glucose is inhibited, thereby increasing blood sugar levels (hyperglycemia). As glucose accumulates in the blood, excess levels of sugar are excreted in the urine (glycosuria). Other symptoms of diabetes include increased urinary volume and frequency, thirst, itching, hunger, weight loss, and weakness.
There are two varieties of the diabetes. Type I is insulin-dependent diabetes mellitus, or IDDM. IDDM was formerly referred to as juvenile onset diabetes. In IDDM, insulin is not secreted by the pancreas and must be provided from an external source. Type II adult-onset diabetes can ordinarily be controlled by diet although in some advanced cases insulin is required.
Before the isolation of insulin in the 1920s, most patients died within a short time after onset. Untreated diabetes leads to ketosis, the accumulation of ketones, products of fat breakdown, in the blood; this is followed by acidosis (accumulation of acid in the blood) with nausea and vomiting. As the toxic products of disordered carbohydrate and fat metabolism continue to build up, the patient goes into diabetic coma.
Treatment of diabetes typically requires regular injections of insulin. The use of insulin as a treatment for diabetes dates to 1922, when Banting et al. ("Pancreatic Extracts in the Treatment of Diabetes Mellitus," Can. Med. Assoc. J., 12, 141-146 (1922)) showed that the active extract from the pancreas had therapeutic effects in diabetic dogs. Treatment of a diabetic patient in that same year with pancreatic extracts resulted in a dramatic, life-saving clinical improvement. Due to the inconvenience of insulin injections, insulin has been the focus of massive efforts to improve its administration and bioassimilation.
The insulin molecule consists of two chains of amino acids linked by disulfide bonds (mw.apprxeq.6,000). The .beta.-cells of the pancreatic islets secrete a single chain precursor of insulin, known as proinsulin. Proteolysis of proinsulin results in removal of four basic amino acids (numbers 31, 32, 64 and 65 in the proinsulin chain: Arg, Arg, Lys, Arg respectively) and the connecting ("C") polypeptide. In the resulting two-chain insulin molecule, the A chain has glycine at the amino terminus, and the B chain has phenylalanine at the amino terminus.
Insulin may exist as a monomer, dimer or a hexamer formed from three of the dimers. The hexamer is coordinated with two Zn.sup.++ atoms. Biological activity resides in the monomer. Although until recently bovine and porcine insulin were used almost exclusively to treat diabetes in humans, numerous variations in insulin between species are known. Porcine insulin is most similar to human insulin, from which it differs only in having an alanine rather than threonine residue at the B-chain C-terminus. Despite these differences most mammalian insulin has comparable specific activity. Until recently animal extracts provided all insulin used for treatment of the disease. The advent of recombinant technology allows commercial scale manufacture of human insulin (e.g., Humulin.TM. insulin, commercially available from Eli Lilly and Company, Indianapolis, Ind.).
The problems associated with oral administration of insulin to achieve euglycemia in diabetic patients are well documented in pharmaceutical and medical literature. Insulin is rapidly degraded by digestive enzymes in the GI tract which results in biologically inactive drug. The membrane permeability is also low due to the lack of sufficient lipophilicity(1). Oral delivery systems that effectively address these two big problems should improve intestinal absorption.
In our prior patents (U.S. Pat. Nos. 5,359,030; 5,438,040; and 5,681,811), we have shown that the amphiphilic modification of insulin improves its lipophilicity and stabilizes it against enzymatic degradation. However, the present inventors have surprisingly discovered insulin conjugates that enable oral delivery, provide delayed onset and/or extended duration of action, as well as dramatically enhancing the activity of insulin.